Exp. Anim. 60(1), 47–56, 2011
—Original—
Effect of Short Period Vasectomy on FSH, LH,
Inhibin and Testosterone Secretions, and
Sperm Motility in Adult Male Rats
Longquan REN1, 2)*, Qiang WENG2, 3)*, Miori KISHIMOTO2), Gen WATANABE1, 2),
Sukanya JAROENPORN4), and Kazuyoshi TAYA1, 2)
Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu
University, Gifu 501-1193, 2)Laboratory of Veterinary Physiology, Department of Veterinary Medicine,
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan,
3)
College of Biological Science and Technology, Beijing Forestry University, Beijing 100083,
China, and 4)Primate Research Unit, Department of Biology, Faculty of Science,
Chulalongkorn University, Bangkok 10330, Thailand
1)
Abstract: The present study was undertaken to clarify changes in secretions of FSH, LH,
inhibin and testosterone, and sperm motility after bilateral vasectomy in adult male rats.
Bilateral vasectomy was created surgically (treated group) and intact rats were used as
control (control group). On days 3, 5, 7, 14, 30, 60, and 90 after surgery, plasma concentrations
of FSH, LH, inhibin, and testosterone were measured by radioimmunoassay, and sperm
motility characteristics were measured by computer-assisted sperm analysis (CASA). The
results show that weights of epididymides significantly increased in vasectomized rats as
compared to control rats. Histologically, damage to spermatogenesis was observed in
vasectomized rats. Multinucleated giant cells were observed in the lumen of some seminiferous
tubules, and there were degenerative spermatids in the epididymides of vasectomized rats.
Plasma levels of LH, FSH, and testosterone only decreased on day 3 after vasectomy;
however, plasma levels of ir-inhibin significantly increased on day 3 after vasectomy. In
addition, the sperm motility parameters, straight-line velocity, curvilinear velocity, deviation
of the sperm head from the mean trajectory and the maximum amplitude of lateral head
displacement were decreased from day 60 after vasectomy. These results suggest that
vasectomy reduces sperm motility starting from day 60 after vasectomy, and early bilateral
vasectomy does not strongly affect the endocrine function of the testis, though it may result
in damage to spermatogenesis in vasectomized rats.
Key words: epididymis, sperm motility, testis, vasectomy
(Received 22 June 2010 / Accepted 6 September 2010)
Address corresponding: K. Taya, Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
*L.R. and Q.W. contributed equally to this study.
48
L. Ren, ET AL.
Introduction
Vasectomy is regarded as the safest method now available for male fertility control. More than 100 million
men worldwide have used vasectomy for family planning
[3]. Vasectomy is also used in the field for controlling
numbers of wild animals. In the field of experimental
animals, vasectomy of males is generally used for inducing pseudopregnancy in females. Following vasectomy,
however, an obstruction is suddenly imposed on a previously patent duct and an inflammatory reaction frequently ensues as sudden backpressure from accumulating
spermatozoa causes the duct to swell and eventually
burst, leading to formation of sperm granulomas [23].
Not surprisingly, >80% of vasectomized men have antisperm antibodies in the peripheral blood within a year
[1]. In a survey on the effects of chronic vasectomy in
laboratory animals (hamster, rat, and monkey), Bedford
[7] observed rapid formation of sperm granulomas in
nearly all cases, either at the site of ligation on the vas
deferens or in the cauda epididymidis. In the vasectomized rats, leakage and granuloma formation invariably
occur about 2 weeks after vasectomy, either at the vasectomy site or in the tail of the epididymis [31, 32,
46].
After leaving the testis, mammalian spermatozoa must
transit through the epididymis to reach the vas deferens
[26]. The mammalian epididymis has two principal
functions. It creates a unique microenvironment within
the lumen of the duct that helps transform immotile, immature testicular spermatozoa into fully fertile competent
cells, and it also stores fertile spermatozoa in a viable
state within the cauda epididymidis/vas deferens regions
until they are ejaculated [23]. Although it is known that
these functions are altered under vasectomy, the different
animal models used to study effects of vasectomy on the
male reproductive tract have given varying results between species and even between individuals [7]. Vasectomized males generally show normal mating behavior;
however, the consequences of vasectomy on the pattern
of reproductive hormones and sperm motility are poorly
documented. To develop knowledge for understanding
reproductive physiology in vasectomized animals, in the
present study, we designed a vasectomized rats model
to monitor sperm motility characteristics using a com-
puter-assisted sperm analysis system (CASA). We also
elucidated the spermatogenic changes caused by vasectomy in adult male rats. In addition, changes in secretions of follicle-stimulating hormone (FSH), luteinizing
hormone (LH), immunoreactive (ir)-inhibin, and testosterone were determined by radioimmunoassay (RIA)
after bilateral vasectomy in adult male rats.
Materials and Methods
Animals and treatments
Adult male Wistar-Imamichi strain rats (Iar: Wistar,
originated from Wistar Institute Standard Strain Lot
No.1359) weighing between 350–450 g, and 3–4 months
of age, were used in this study. Animals were anesthetized by ether and subjected to vasectomy by transection
of a U-shaped segment from the vas deferens between 2
ligatures. Sham-operated controls underwent the same
surgical procedure without vasectomy. Rats were returned to their room, under a light regimen of 14 h
light/10 h dark, and were given water and commercial
food ad libitum. Care and use of the animals were in
accordance with the requirements established under the
Guide for the Care and Use of the Laboratory Animals
by the Tokyo University of Agriculture and Technology,
Japan.
Sample collection
Rats were decapitated 3, 5, 7, 14, 30, 60, and 90 days
postoperation. Blood samples were collected and both
testes and epididymis were removed. Blood samples
were collected from each animal into individual heparinized centrifuge tubes, and plasma samples were immediately obtained by centrifugation at 1,700 × g for 15
min at 4°C. Plasma samples were stored at –20°C until
assayed for FSH, LH, ir-inhibin, and testosterone. Testes and epididymis were fixed in 4% paraformaldehyde
in 0.1 M PBS for morphological observation.
Sperm concentration
The concentration of spermatozoa in the testes was
examined by a method described previously [34]. In
brief, tunica albuginea was removed from the testes,
which were then homogenized in 10 ml saline by a homogenizer (Physcotron; Microtech Nition, Chiba, Japan)
49
vasectomy in adult MALE rats
for 10 s, followed by sonication using a sonicator
(TOMY, SEIKO Co., Ltd., Tokyo, Japan) for 3 min on
ice. One drop of extracted emulsion was placed on a
hemocytometer after adequate dilution with saline, and
sperm heads were then counted under a phase contrast
microscope. The remaining extracts were centrifuged
at 38,000 × g for 30 min at 4°C, and supernatants were
stored at –20°C for hormone assays.
Sperm motility analysis
Semen from the cauda epididymidis was collected into
1.5 ml tubes containing 1 ml of modified Tyrode’s medium (3 μl semen sample from the cauda epididymidis
diluted with 1 ml modified Tyrode’s medium). The
sperm motility was measured by CASA using a C.IMAGING C.MEN system. Briefly, diluted sperm suspensions
were placed in prewarmed slide chambers with depths
of 20 μm. The slides were viewed using an Olympus
microscope (Olympus BX50F, Olympus optical Co.,
Tokyo, Japan) equipped with a 4 × dark field optics and
a video camera (CCD XC77, Sony Co., Tokyo, Japan)
connected to a personal computer. The temperature of
the microscope stage was maintained at 37°C throughout
the observation by a stage warmer (MP-10DM, Kitazato Supply Co., Shizuoka, Japan). CASA was performed
using the C.IMAGING C.MEN system and C.IMAGING
software (C.IMAGING systems, Compix Inc., Tualatin,
OR, USA). Our CASA system analyzes 15 consecutive,
digitalized photographic images obtained from a single
field. These 15 consecutive photographs were taken with
a time lapse of 0.5 s. Two to three separate fields were
taken for each sample. Percentage of motile spermatozoa (%), straight-line velocity (VSL, μm/s), curvilinear
velocity (VCL, μm/s), linearity (ratio of the straight line
distance to the actual tracked distance), deviation of the
sperm head from the mean trajectory (ALH, mean μm)
and the maximum amplitude of lateral head displacement
(ALH, max μm) and beat frequency of centroids crossing
the average trajectory (BCF, Hz) were determined.
Histology
Fixed testes and epididymides were dehydrated
through a series of graded concentrations of ethanol and
xylene, and embedded in paraffin. The paraffin-embedded testes were serially sectioned at 4–6 μm thickness
and placed on poly-L-lysine coated slide glasses. The
sections were stained with hematoxylin-eosin (HE) for
morphological observations.
RIA for FSH, LH, ir-inhibin, and testosterone
Concentrations of FSH, LH, ir-inhibin, and testosterone in the plasma were determined by specific RIAs.
Iodinated preparations were rat FSH-I-7 and LH-I-7.
The antisera used were anti-rat FSH-S-11 and LH-S-10.
Results were expressed in terms of NIDDK rat FSHRP-2 and LH-RP-2. The intra-assay and inter-assay
coefficients of variation were 3.4 and 5.3% for FSH and
7.2 and 11.2% for LH, respectively. Plasma concentrations of ir-inhibin were measured as described previously [20]. The iodinated preparation was 32 kDa bovine
inhibin and the antiserum used was rabbit antiserum
against bovine inhibin (TNDH-1). Results were expressed in terms of 32 kDa bovine inhibin. The intra- and
inter-assay coefficients of variation were 8.8 and 14.4%,
respectively. Testicular contents and plasma concentrations of testosterone were determined by a double-antibody RIA system with 125I-labeled radioligands as described previously [49]. The antiserum against
testosterone (GDN 250) was kindly provided by Dr. G.
D. Niswender (Colorado State University, Fort Collins,
CO, USA). The intra- and inter-assay coefficients of
variation were 6.3 and 7.2%, respectively.
Statistical analysis
All data are expressed as means ± SEM of five animals. One-way ANOVA was performed, and the significance between two means was determined by Student’s t-test, A value of P<0.05 was considered
statistically significant.
Results
Weights of testis, epididymis, prostate, and seminal
vesicle
The weights of epididymides in the vasectomy group
showed a significant increase until 30 days postoperation
compared with control group (Fig. 1B). However, the
weights of epididymides decreased from 30 days postoperation compared with the control group (Fig. 1B). In
addition, there were no significant changes in the weights
50
L. REN, ET AL.
Fig. 1. Weights of testes (A), epididymis (B), prostate (C), and seminal vesicle and coagulating gland complex (SV+CG) (D) of rats after vasectomy (solid bar) or control (open bar). Each point represents the mean ± SEM of five animals. *P<0.05
compared to control value.
of testes, prostate, and seminal vesicles between the
vasectomy and control groups (Fig. 1A, 1C, and 1d).
Sperm concentrations
data from the sperm head count are shown in Fig. 2.
There was no significant difference between the vasectomy and control groups in sperm head count.
Morphology of testes and epididymis
Morphology of testes and epididymides of the rats
after vasectomy are shown in Fig. 3. No changes of
morphology of testes or epididymides were observed in
the control from 3 to 90 (Fig. 3A and 3E). Only four of
35 vasectomized rats showed connection between the
epididymal duct and a sperm granuloma through which
spermatozoa appeared to be escaping (Fig. 3B and 3C).
These four rats had abnormal testes. damage to sper-
Fig. 2. Sperm head count in testes after induced vasectomy
(solid bar) or control (open bar). Each point represents
the mean ± SEM of five animals.
51
VASECTOMY IN AdULT MALE RATS
Sperm motility
The sperm of the four rats which formed sperm granulomas after vasectomy did not move. data from CASA
are shown in Fig. 4. In the vasectomy group, the sperm
motility parameters, straight-line velocity (the average
velocity measured in a straight line from the beginning
to the end of the track, µm/s), curvilinear velocity (the
average velocity measured over the actual point to point
track, µm/s), ALH mean and ALH max began to decrease
60 days after vasectomy. (Fig. 4A–d). There were no
significant changes in three sperm motility parameters,
the mean percentage of sperm motility (%), BCF (Hz),
and linearity index after vasectomy (Fig. 4E–G).
Fig. 3. Morphological changes of the epididymis and testis of
adult male rats 7 days after vasectomy. (A and E)
Epididymis and testes control. (B and C) Profile of an
epididymal duct leacking into a sperm granuloma. (d)
The epididymal duct is filled with degenerated spermatogenic cells. (F) Multinucleated giant cells (arrows) and
round spermatids and elongated spermatids were massively lost into the lumen of seminiferous tubules. Ed:
epididymal duct, SG: sperm granuloma, ST: seminiferous
tubule, R: sloughed round spermatids. Staining: hematoxylin and eosin. Scale bars: A and C, 100 µm; B, 200
µm; d, 50 µm.
matogenesis occurred in vasectomized testes at days 7
and 14, but from 1 to 3 months the damage was not found
in vasectomized testes. A few multinucleated giant cells
could be seen in the lumen of some seminiferous tubules
and their characteristics were similar to those of abnormal isolated round spermatids (Fig. 3F). Sertoli cells
seemed unaffected. In addition, round spermatids and
elongated spermatids were massively lost into the lumen
of the seminiferous tubules (Fig. 3F). The granulomas
that were receiving spermatozoa from the epididymal
duct were of the epididymis despite the presence of
granulomas closer to the testis. On the other hand, round
degenerated spermatids or spermatozoa were found
within the epididymal duct (Fig. 3d).
Plasma concentrations of LH, FSH, testosterone, and
ir-inhibin
Plasma concentrations of LH were significantly lower in the vasectomized group on day 3 after the operation
than the control group, then increased gradually until
day 7, and became significantly higher than that of the
control group on day 90 after the operation (Fig. 5A).
There were no differences in plasma concentrations of
FSH between vasectomized and control rats (Fig. 5B).
There was a significant decrease in testosterone concentrations on day 3 postoperation in vasectomized rats, but
they increased again to become comparable to control
levels (Fig. 5C). Plasma concentrations of ir-inhibin
increased remarkably, after day 3 in the vasectomized
group compared with the control group; however, inhibin levels decreased in the vasectomized group compared to the control level (Fig. 5d).
Discussion
The present results demonstrate that short period vasectomy in adult rats significantly increased epididymis
weights, induced morphological changes in seminiferous
tubules and epididymal ducts, and decreased sperm motility parameters. However, apart from day 3, there were
no significant changes in plasma concentrations of LH,
FSH, testosterone, and ir-inhibin after vasectomy. These
results suggest that vasectomy in rats leads to early damage to spermatogenesis and decreased sperm motility;
however, vasectomy did not strongly affect the endocrine
function of the testis in adult male rats.
52
L. REN, ET AL.
Fig. 4. Changes in epididymal sperm motility parameters: straight velocity (A), curvilinear velocity (B), amplitude of lateral displacement (ALH) mean (C), amplitude
of lateral displacement (ALH) max (D), percentages of motile spermatozoa (E),
linearity index (F), and beat frequency of centroids crossing the average trajectory (BCF, Hz) (G) after vasectomy (solid bar) or control (open bar). Each point
represents the mean ± SEM of five animals. *P<0.05 compared to control
group.
VASECTOMY IN AdULT MALE RATS
53
Fig. 5. Changes in plasma concentrations of (A) luteinizing hormone (LH), (B) follicle-stimulating hormone (FSH), (C) testosterone, and (d) immunoreactive (ir-) inhibin in male rats
after vasectomy (solid bar) or sham operation (open bar) in rats. Each value represents the
mean ± SEM of five animals. *P<0.05 compared to control value.
Common features can be discerned in the response of
the epididymis to vasectomy, despite species differences. Increases in size and number of lysosomes are
the most frequent changes in the epididymal epithelium
[17]. In the present study, the weight of epididymides
in the vasectomy group showed a rapid increase starting
on day 3, but there were no significant differences in the
weights of testes, prostate and seminal vesicles between
the vasectomy and control groups. The change of
epididymal weight may be due to accumulation of spermatozoa in the epididymis [22, 41]. Studies of vasectomized rams have shown that the seasonal pattern was
ill-defined, and the testicular parameters were not only
lower, but the cauda epididymidis was larger than in
intact rams [41]. In this respect, the rabbit has proved a
better experimental model as the vas deferens and cauda
epididymidis are quite distensible and, following vasectomy, can accommodate considerable backpressure from
accumulated fluid and spermatozoa without rupturing
[7, 22]. Also, Moore and Bedford [35] observed a linear
increase in the number of spermatozoa in the epididymides
of rabbits vasectomized for up to 6 months that equated
closely with estimates of sperm production by the testes.
In the present study, though there was no significant
changes in the weights of testes, multinucleated giant
cells were found in the lumen of some seminiferous tubules in vasectomized rats. Multinucleated giant cells
are common in vasectomy-induced spermatogenic damage, probably as a result of germ cell sloughing, an increase of pressure in the seminiferous tubules and an
autoimmune reaction [37, 47, 51]. A histophysiological
study of vasectomized rats indicated that a higher frequency of stage Ⅶ–Ⅷ of the tubular cycle was observed, showing sperm accumulation [25]. At 5 and 10
weeks after vasectomy, vasectomized left testes were
significantly lighter than unvasectomized right testes and
sham-operated testes. During that period, the seminiferous tubules of vasectomized testes were highly damaged,
presenting narrow tubular diameter, disorder of cellular
54
L. Ren, ET AL.
arrangement, depletion of germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per
cross-sectional area than sham-operated testes at 5 and
10 weeks after operation [24]. The damage is probably
pressure-mediated rather than immunological [30], because dramatic damage occurred in a short period after
vasectomy.
There were significant decreases in plasma levels of
LH, FSH, and testosterone only on day 3 after vasectomy. In previous studies, surgical [2, 29, 36, 50],
physical [11, 13, 28, 38, 39], and social stress [43–45]
resulted in decreased plasma levels of gonadotropins,
inhibin and/or testosterone in humans, male primates
and rats. However, in the present study, plasma levels
of inhibin significantly increased on day 3 after vasectomy. The increase in plasma levels of inhibin may be
similar to efferent duct ligation indicating that the inhibin secreted into the seminiferous tubule lumen is
trapped [4]. However, the accumulated inhibin did not
appear to influence FSH secretion significantly, probably
because of the integrity of the blood-test barrier and the
failure of the raised intratubular levels to affect an increase in the secretion of inhibin across the base of the
Sertoli cells into testicular lymphatics [5]. Nevertheless,
others have suggested that the passage of inhibin into
the rete testis fluid and its subsequent absorption represents an important pathway by which inhibin reaches the
circulation [27].
The percentage of progressively motile spermatozoa
and sperm motion parameters were recorded in the present study by CASA, which is more objective than visual assessment. There were significant differences
between vasectomized and control rats in the VSL, VCL,
ALH mean, and ALH max from 60 days after operation.
In many studies [15, 16, 21, 55], a correlation between
sperm motility and fertility has been demonstrated.
Sperm motion parameters are important for oocyte penetration; progressive sperm motility is essential for efficient penetration. The characterization and regulation
of the epididymal protein microenvironment has been
of interest for many years [10, 48, 54], and it has been
established that some of these proteins are important for
sperm maturation and sperm function at the site of fertilization. Vasectomy significantly reduces concentra-
tions of the major protein, cysteine-rich secretory protein
(CRISP-1), and the minor proteins, phosphatidylethanolamine-binding protein and prostaglandin D2 [52, 53].
Furthermore, there is a significant increase in antisperm
antibody production after vasectomy [12]. The main risk
factor for the development of antisperm antibodies in
the male is disruption of the vas deferens, which is
achieved during vasoresection for sterilization. Antisperm antibodies in men occur in blood and seminal
fluid, and attach to the sperm surface [33]. Antisperm
antibodies may have heterogeneous effects, depending
on their cognate antigens [40]. They have been found
to affect sperm motility [6, 19], the acrosome reaction
[8], penetration of cervical mucus [14], sperm binding
to the zona pellucida [18], and sperm-egg fusion [9, 42].
In addition, in the present study, sperm granulomas were
histologically observed in 4 of 35 rats, and degenerated
spermatids were found within the lumen of the cauda
epididymis. Taken together, the results of the present
study suggest that early bilateral vasectomy in the rats
may affect sperm maturation and motility.
In conclusion, the present results demonstrate that
early bilateral vasectomy decreased sperm motility, and
damaged spermatogenesis in vasectomized testes, as well
as inducing formation of sperm granulomas and degenerative spermatozoa in the epididymis. However, the
present results also provide new evidence that early bilateral vasectomy does not affect Sertoli cells and the
endocrine function of the testis in vasectomized rats.
Acknowledgments
We are grateful to the National Hormone and Peptide
Program, Harbor-UCLA Medical Center, Torrance, CA,
USA and Dr. A. F. Parlow for providing RIA materials
of rat LH and FSH. We are also grateful to Dr. G. D.
Niswender (Animal Reproduction and Biotechnology
Laboratory, Colorado State University, Fort Collins, CO,
USA) for the generous gift of antiserum to testosterone
(GDN 250). This work was partially supported by a
JSPS Research Fellowship for Young Scientists (14.
20179), a Grant-in-Aid for Scientific Research (No.
03338) from the Japan Society for the Promotion of Science and Program for Changjiang Scholars and Innovative
Research Team in Universities (IRT0607) of China.
vasectomy in adult MALE rats
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